Purification and analysis of molecules is very often carried out by forcing these molecules to migrate through a gel. In gel electrophoresis the driving force is a voltage gradient across the gel and the gel matrix comprises natural or synthetic polymers. The synthetic polymers are usually formed by polymerization of double bonds present in monomer and cross-linker molecules. The monomers currently in use are of the amide type and they include acrylamide, N-acryloyl-tris(hydroxymethyl)aminomethane (NAT) (references 1-3) and N-acryloyl morpholine (reference 4). The gels made of the last two monomers have been recently introduced. The most important features of poly(NAT) gels are pronounced hydrophilicity and a higher porosity compared to polyacrylamide gels. The higher porosity of poly(NAT) gels could be advantageously used for separation of larger molecules by electrophoresis and isoelectric focusing (references 1-3). It appeared worth searching for a monomer which will produce even more porous gel, since such a gel would be beneficial in many applications, including isoelectric focusing, multiphasic zone electrophoresis and electrophoresis of proteins, lipoproteins, proteoglycans and nucleic acids. As a working hypothesis it was assumed that NAT yields gels of higher porosity because its molecular weight is higher than that of acrylamide. Thus, an NAT solution has a molar concentration lower than the acrylamide solution of the same percentage. After polymerization the lower molar concentration of the NAT solution presumable results in fewer polymer chains per unit volume, leading to gels of increased porosity. The finding (reference 1) that a poly(NAT) gradient gel exhibited a porosity approximately three-fold higher than porosity of the corresponding polyacrylamide gel, in accordance with the 2.5 fold lower molarity, lend support to the above assumption. If this simple assumption is correct, then even more porous gels will be formed from monomers of higher molecular weight. In addition to size of a monomer, other factors can influence porosity of a gel. Thus, if interactions exist between monomer molecules, or between a monomer and a growing polymer, or between the growing polymer chains, then as a result of these interaction the polymer chains may not be randomly distributed. If they form some kind of bundles, larger pores will be created. In addition to a higher molecular weight, the new monomers should fulfill at least two further requirements. First, they should be hydrophilic in order to give homogenous aqueous gels. Second, the double bond of these monomers should efficiently polymerize under rather mild conditions used for the preparation of gels for electrophoresis. From the above assumptions and considerations, it appeared that gels with desirable properties may be formed of monomers derived from amino sugar alcohols, as described below by the formula: ##STR2## where R.sub.1 is H, CH.sub.2 OH or (CHOH).sub.m CH.sub.2 OH, m being 1 or 2;
R.sub.2 is H or CH.sub.3 ; PA1 R.sub.3 is H or CH.sub.3 ; and PA1 n is an integer of 1-4; PA1 R.sub.2 is H, a monohydroxy alkyl, a polyhydroxy alkyl or a hydrocarbon radical, preferably of 1 to about 30 carbon atoms; PA1 R.sub.3 is H or CH.sub.3 ; and PA1 n is an integer of 1-4;
These monomers are hydrophilic as they contain at least three hydroxyl groups. Further, due to adjacent amide group the double bond in the monomers is expectedly more reactive than a typical double bond.
Two of the monomers represented by the above formula, N-acryloyl-1-amino-1-deoxy-D-glucitol and N-methacryloyl-1-amino-1-deoxy-D-glucitol as well as their linear polymers are known (reference 5 and 6). However, in the two references no data were reported concerning polymerization of either of the two monomers in the presence of a cross-linker to form an aqueous gel. Moreover, there was no indication as to whether such an aqueous gel may represent a matrix suitable for electrophoretic separation of molecules.
The monomers of the above formula include compounds with the nitrogen atom linked to a carbon atom having one or two hydrogens as well as the compounds in which hydrogen from the amide nitrogen is substituted by methyl group. In the two known monomers, N-acryloyl-1-amino-1-deoxy-D-glucitol and N-methacryloyl-1-amino-1-deoxy-D-glucitol, the nitrogen is linked to a carbon atom with two hydrogens. The attempts to synthesize other monomers of the formula above by the process described in reference 5 failed. We have initially used sugar precursors readily available, that is N-acetyl-2-amino-2-deoxy-D-glucose and N-methyl-1-amino-1-deoxy-D-glucitol. However, neither N-acryloyl-2-amino-2-deoxy-D-glucitol, a compound with the nitrogen atom linked to carbon with one hydrogen, nor N-methyl-N-acryloyl-1-amino-1-deoxy-D-glucitol, a compound with methyl group on the nitrogen, crystallized from the water-ethanol-ether solution of reference 5. After modification of the preparation process, as described in detail in reference 7, all monomers of the general formula could be obtained. The modified process includes treatment of the reaction mixture with an anionic and a cationic ion exchanger to remove the remaining reactants and formed byproducts. Further, the water solution of the monomer is evaporated at atmospheric pressure. Those monomers which dried out to give a solid residue were recrystallized and those remaining as a viscous liquid were directly used for preparation of gels.
As shown in reference 7, the new monomers could be polymerized in presence of a cross-linker to give water insoluble gels. It was shown that such a gel is a matrix suitable for isoelectric focusing and therefore also for other electrophoretic techniques, since it is well known that requirements imposed on a matrix for isoelectric focusing are more stringent than those for other electrophoretic techniques. For example, agarose needs to be modified in a special way before it is suitable for preparation of isoelectric focusing gels (references 8 and 9).
Gels made of the monomers of the general formula are suitable for electrophoresis, as demonstrated by experimental results of reference 7 in accordance with the assumptions discussed above. However, it is not apparent whether there may be important differences in properties of gels made of various monomers of the general formula. Thus, if molecular weight of the monomer determines porosity of the gel, then gels prepared from equal amounts of monomers having identical molecular weight should exhibit equal porosity. In that case, the gels made for example of N-acryloyl-1-amino-1-deoxy-D-glucitol,N-acryloyl-2-amino-2-deoxy-D-glucito l and N-acryloyl-1-amino-1-deoxy-D-galacitol will be equally porous. On the other hand, if gel formation differs from one monomer to the other because the polymerization rate of each monomer is unique or if polymer interactions are different, then the resulting porosity will not be equal. Since our current knowledge on porosity of synthetic gels is based on only three monomers, that is acrylamide, NAT and N-acryloyl morpholine, which are structurally more different than the monomers of this invention, it was not possible to make predictions about porosity of gels made of structurally related monomers including isomers.
Electrophoretic migration of molecules in polyacrylamide gels is mostly described in terms of the extended Ogston model (references 10 and 11). Accordingly, the measured mobility, .mu., can be related to the free mobility, .mu..sub.o, of a migrating molecule with radius R, as well as to the gel percentage T, total length of the gel fibers, l', and the fiber radius, r: EQU log .mu.=log .mu..sub.o -.pi.l'(r+R).sup.2 T.times.10.sup.-16 EQU or EQU log .mu.=log .mu..sub.o -K.sub.r T
where the retardation coefficient, K.sub.r, is defined as EQU K.sub.r =.pi.l'(r+R).sup.2 .times.10.sup.-16
The extended Ogston model has been extensively used to analyze electrophoretic migration of various macromolecules in polyacrylamide gels, mainly in order to enable estimation of the molecular weight and radius of an unknown molecule. However, since the retardation coefficient is correlated also to the length of gel fibers and their radius, this model can be used as an approach for characterization of different gels. After analysis of gels made of the monomers shown by the general formula above according to the extended Ogston model, it was surprisingly found that gel properties vary significantly from one gel to the another, as described in this invention.